Development and validation of a sensitive LC-MS-MS method to quantify psilocin in authentic oral fluid samples.

Psilocin is an active substance and a dephosphorylated product of psilocybin formed after the ingestion of mushrooms. The low stability caused by the quick oxidation of this analyte requires sensitive methods for its determination in biological matrices. In this work, we described the development, optimization and validation of a method for the quantification of psilocin in authentic oral fluid samples by liquid chromatography-tandem mass spectrometry. Liquid-liquid extraction was performed using 100 µL of oral fluid samples collected with a Quantisal™ device and t-butyl methyl ether as the extraction solvent. The method showed acceptable performance, with limits of detection and quantification of 0.05 ng/mL, and the calibration model was achieved between 0.05 and 10 ng/mL. Bias and imprecision results were below -14.2% and 10.7%, respectively. Ionization suppression/enhancement was lower than -30.5%, and recovery was >54.5%. Dilution integrity bias was <14.4%. No endogenous and exogenous interferences were observed upon analyzing oral fluid from 10 different sources and 56 pharmaceuticals and drugs of abuse, respectively. No carryover was observed at 10 ng/mL. Psilocin was stable in oral fluid at -20°C, 4°C and 24°C up to 24, 72 and 24 h, respectively, with variations <17.7%. The analyte was not stable after three freeze/thaw cycles, with variations between -73% and -60%. This suggests the instability of psilocin in oral fluid samples, which requires timely analysis, as soon as possible after the collection. The analyte remained stable in processed samples in an autosampler (at 10°C) for up to 18 h. The method was successfully applied for the quantification of five authentic samples collected from volunteers attending parties and electronic music festivals. Psilocin concentrations ranged from 0.08 to 36.4 ng/mL. This is the first work to report psilocin concentrations in authentic oral fluid samples.

High-sensitivity method for the determination of LSD and 2-oxo-3-hydroxy-LSD in oral fluid by liquid chromatography‒tandem mass spectrometry.

PURPOSE: We have developed and validated a high-sensitivity method to quantify lysergic acid diethylamide (LSD) and 2-oxo-3-hydroxy-LSD (OH-LSD) in oral fluid samples using liquid-liquid extraction and liquid chromatography-tandem mass spectrometry (LC‒MS/MS). The method was applied to the quantification of both substances in 42 authentic oral fluid samples. METHODS: A liquid-liquid extraction was performed using 500 µL each of samples (oral fluid samples collected using Quantisal™ device) and dichloromethane/isopropanol mixture (1:1, v/v). Enzymatic hydrolysis was evaluated to cleave glucuronide metabolites. RESULTS: The limit of quantification was 0.01 ng/mL for both LSD and OH-LSD. The linearity was assessed between 0.01 and 5 ng/mL. Imprecision and bias were not higher than 10.2% for both analytes. Extraction recovery was higher than 69%. The analytes were stable in the autosampler at 10 °C for 24 h, and up to 30 days at 4 and -20 °C. The method was applied to the analysis of 42 oral fluid samples. LSD was detected in all samples (concentrations between 0.02 and 175 ng/mL), and OH-LSD was detected in 20 samples (concentrations between 0.01 and 1.53 ng/mL). CONCLUSIONS: A high-sensitive method was fully validated and applied to authentic samples. To our knowledge, this is the first work to report concentrations of LSD and OH-LSD in authentic oral fluid samples.

Comparison of Cocaine/Crack Biomarkers Concentrations in Oral Fluid, Urine and Plasma Simultaneously Collected From Drug Users.

The use of oral fluid (OF) as an alternative specimen for drug analysis has become very popular in forensic toxicology. Many clinical studies have evaluated the correlations between concentrations of cocaine and its metabolites in OF and other matrices, but results have shown high variability. In addition, there are no data available regarding the correlations between biomarkers of crack-cocaine use in different matrices. This study evaluated the relationship between concentrations of cocaine/crack-cocaine biomarkers in OF, urine and plasma samples collected from cocaine users. All samples were analyzed for the presence of cocaine (COC), benzoylecgonine (BZE) and anhydroecgonine (AEC) by a validated liquid chromatography-mass spectrometry method. Median COC, BZE and AEC concentrations ranged from 4.20 to 33.26 ng/mL, from 13.03 to 3,615.86 ng/mL and from 7.40 to 1,892.5 ng/mL across matrices, respectively. The relationship between drug concentrations in OF versus plasma (OF/P) and OF versus urine (OF/U) was evaluated by their coefficients of determination (R2). Least-squares regression analyses demonstrated significant correlations between OF/P and OF/U for cocaine and BE (P < 0.05), with R2 = 0.17, 0.07 for cocaine and R2 = 0.73, 0.45 for BE, respectively. The correlation coefficients (r) found for BZE, COC and AEC in OF/P and OF/U were 0.85 and 0.67 (P < 0.05); 0.41 and 0.26 (P < 0.05); and 0.30 and -0.37 (P > 0.05), respectively. Many factors contribute to the variability of drug correlation ratios in studies involving random samples, including uncertainty about the time of last administration and dosage. Overall, we found significant R2 values for COC and BZE in OF/P and OF/U, but not for AEC. Despite the good correlations found in some cases, especially for BZE, the large variation in drug concentrations seen in this work suggests that OF concentrations should not be used to estimate concentrations of COC, BZE or AEC in plasma and/or urine.

Oral Fluid Testing for Cocaine: Analytical Evaluation of Two Point-of-Collection Drug Screening Devices.

The use of point-of-collection testing (POCT) devices for drugs of abuse in oral fluid (OF) is an advantageous tool that has been used for different purposes-particularly traffic enforcement. However, even with the widespread report of cocaine consumption, the reliability of POCT devices has been reported in different magnitudes. This study evaluated the reliability of two POCT devices for the detection of cocaine in OF samples of 110 cocaine users: (i) the DDS2™ (cutoff = 30 ng/mL) and (ii) the Multi-Drugs Multi-Line-Twist Screen Test Device™ (MDML) (cutoff = 20 ng/mL). Results of the screening tests were compared with a Liquid Chromatography-Mass Spectrometry (LC-MS) assay. Sensitivity, specificity and accuracy of DDS2™ were 100, 77.77 and 80% when compared with LC-MS with a cutoff of 30 ng/mL, and 88.89, 89.15 and 89.09% with a cutoff of 10 ng/mL. The MDML™ device achieved sensitivity, specificity and accuracy of 100, 65.6 and 70.9% when compared with LC-MS with a cutoff of 20 ng/mL, and 92.6, 71.1 and 76.6% with a cutoff of 10 ng/mL. When compared with a 10 ng/mL cutoff, the DDS2™ achieved reliability parameters higher than 80%. On the other hand, the MDML™ device did not achieve the minimal recommendation of 80% for all parameters at the same time. Taking into consideration the reliability results showed here, the authors believe that the use of these POCT devices seems to be suitable for cocaine detection in forensic tests only if all positive specimens are further confirmed by a validated method.

Multianalytical Method Validation for Qualitative and Quantitative Analysis of Solvents of Abuse in Oral Fluid by HS-GC/MS.

The use of oral fluid as a biological matrix to monitor the use of drugs of abuse is a global trend because it presents several advantages and good correlation to the blood level. Thus, the present work aimed to develop and validate an analytical method for quantification and detection of solvents used as inhalants of abuse in oral fluid (OF), using Quantisal™ as collector device by headspace and gas chromatography coupled with a mass detector (HS-GC/MS). Chromatographic separation was performed with a ZB-BAC1 column and the total time of analysis was 11.8 min. The method showed good linearity (correlation coefficient higher than 0.99 for all solvents). The limits of detection ranged from 0.05 to 5 mg/L, while the lower limits of quantification ranged from 2.5 to 12.5 mg/L. Accuracy, precision, matrix effect, and residual effect presented satisfactory results, meeting the criteria accepted for the validation of bioanalytical methods. The method showed good selectivity considering that, for solvents coeluting at the same retention time, resolution was performed by the mass detector. The method developed proved to be adequate when applied in OF samples from users of drugs and may be used to monitor the abuse of inhalants in routine forensic analyses.